guanosine-triphosphate and Neoplasm-Metastasis

The bisphosphonates (BPs) are well established as the treatments of choice for disorders of excessive bone resorption, including Paget's disease of bone, myeloma and bone metastases, and osteoporosis. There is considerable new knowledge about how BPs work. Their classical pharmacological effects appear to result from two key properties: their affinity for bone mineral and their inhibitory effects on osteoclasts. Mineral binding affinities differ among the clinically used BPs and may influence their differential distribution within bone, their biological potency, and their duration of action. The inhibitory effects of the nitrogen-containing BPs (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their inhibition of farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids used for the posttranslational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular pathways, such as preventing apoptosis in osteocytes, are emerging as other potentially important mechanisms of action. As a class, BPs share several common properties. However, as with other classes of drugs, there are obvious chemical, biochemical, and pharmacological differences among the various individual BPs. Each BP has a unique profile that may help to explain potential important clinical differences among the BPs, in terms of speed of onset of fracture reduction, antifracture efficacy at different skeletal sites, and the degree and duration of suppression of bone turnover. As we approach the 40th anniversary of the discovery of their biological effects, there remain further opportunities for using their properties for medical purposes.

Topics: Animals; Bone and Bones; Bone Neoplasms; Bone Resorption; Diphosphonates; Guanosine Triphosphate; Humans; Models, Biological; Models, Chemical; Multiple Myeloma; Neoplasm Metastasis; Nitrogen; Osteoclasts; Osteocytes; Osteoporosis; Protein Processing, Post-Translational; T-Lymphocytes; Treatment Outcome

Unfortunately, the anticancer drugs that are used nowadays in the clinic have only limited success. To provide a significant clinical advancement, new concepts have to be introduced to aid the design of new tools for therapy. Cancer is not only restricted to neoplastic cells, but rather it involves an ensemble of protagonists. In addition, the evolution of cancer is extremely complex, since multiple cellular activities are involved. Some key steps in the evolution to a metastatic tumor have been shown to be no useful targets. Targeting the stroma cells, however, could bring a new efficiency in anticancer treatment. Targeting the disorganized tissue architecture at the primary site and the restoration of the cell death program in cancer cells appears to create new possibilities in drug design. Also the cytoskeleton, which represents a dynamic set due to its plasticity and multiplicity, seems to be a promising target in anticancer therapy. Moreover, the evolving knowledge of the role of metastasis suppressor genes in regulating cancer cell growth at the secondary site suggests that they could serve as new targets for therapeutic intervention. This review intends to highlight the unraveling of new therapeutic pathways, and to unveil new powerful research tools for combating metastasis.

Topics: Animals; Anoikis; Antineoplastic Agents; Cytoskeleton; Genes, Tumor Suppressor; Guanosine Triphosphate; Humans; Microtubules; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphoinositide-3 Kinase Inhibitors; Polysaccharides; Signal Transduction

Rac GTPases are crucial signaling regulators in eukaryotic cells, acting downstream of many cell surface receptors. They play essential roles in diverse cellular functions including cytoskeleton dynamics, cell motility, cell survival and apoptosis. Their activities are controlled by a tightly regulated GDP/GTP cycle coupled with an alternation between cytoplasm and membrane compartments. Aberrant Rac signaling is found in some human cancers as a result of changes in the GTPase itself or in its regulation loops. This review highlights recent findings regarding the molecular and functional aspects of Rac that mediate tumorigenic transformation and metastasis. It also describes the cellular mechanisms that potentially explain the complex role of Rac in tumorigenesis. Finally, it discusses approaches for modulating Rac function as a potential anticancer strategy.

Topics: Animals; Antineoplastic Agents; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Mice; Mice, Knockout; NADPH Oxidases; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; rac GTP-Binding Proteins

As a result of its transforming abilities, activated Ras is expressed in a great number of cancers. The ras mutation frequency varies between 95% in pancreatic cancer and 5% in breast cancer. In leukemia, the highest frequency (30%) is found in acute myeloid leukemia. The presence of ras mutations has been correlated with a poor prognosis and negative clinical outcome. This suggests that mutated Ras activates mechanisms, which favor tumor growth, enhance the metastatic capacity of tumors or modulate tumor-specific immune responses. Several new functions of Ras, such as downregulation of major histocompatibility complex molecules, upregulation of certain cytokines, growth factors and degradative enzymes have been uncovered in the last decade. Additionally, mutated Ras can also serve as a primary target for the development of immunotherapy or drug therapy. This review will discuss the mechanisms by which Ras expressing tumors are able to evade destruction by the immune system and enhance their growth and metastatic potential. It will further elaborate on the attempts to develop successful immunotherapy and drug therapy targeting Ras expressing tumors.

Topics: Alkyl and Aryl Transferases; Animals; Antigen Presentation; Antineoplastic Agents; Cell Adhesion Molecules; Cell Transformation, Neoplastic; Cytokines; Drug Design; Endopeptidases; Enzyme Activation; Farnesyltranstransferase; Fungal Proteins; Fusion Proteins, bcr-abl; Genes, ras; Growth Substances; Guanosine Triphosphate; Humans; Immune System; Immunotherapy; Leukemia; Mice; Models, Biological; Mutation; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neurofibromin 1; Oligonucleotides, Antisense; Proteins; Proto-Oncogene Proteins p21(ras); Reoviridae Infections; Repressor Proteins; Signal Transduction; SOS1 Protein; T-Lymphocyte Subsets

It is now established that ras oncogenes can induce metastatic characteristics in primary diploid fibroblasts, nonsenescing fibroblasts and nonmetastasizing tumors. The issue of whether ras is directly involved in maintaining the metastatic phenotype through the expression and action of its gene product has been examined by analyzing the relationship to ras expression and to the production of the p21 ras-GTP complex, which is thought to mediate ras-transforming activity. While these expression and mutation studies support the idea that p21 ras directly regulates metastasis formation, it is also evident that there are many examples of human and murine cancers which show no differences in ras expression in primary and metastatic tumor cells. This may be partially explained by the ability of protein kinase-encoding oncogenes to also induce metastatic potential. In addition, the ability of ras to induce metastasis may be dependent on the regulation of its activity by other genes. Furthermore, transformation does not occur as an isolated genetic event, but is rather the result of interaction of two or more oncogenes. We suggest that the nature of these gene interactions will ultimately determine whether a cell is a benign transformant or a malignant and metastatic cancer.

Topics: Animals; Cell Transformation, Neoplastic; Gene Expression; Genes, ras; Guanosine Triphosphate; Humans; Neoplasm Metastasis; Oncogene Protein p21(ras); Oncogenes

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death in the world. Inhibitor of differentiation 1 (Id1) is overexpressed in NSCLC and involved in promoting its progression and metastasis. Identifying natural compounds targeting Id1 may have utility in NSCLC treatment. Here, we sought to determine whether the anti-tumor activities of

Topics: A549 Cells; alpha7 Nicotinic Acetylcholine Receptor; Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Flavanones; Flavonoids; Guanosine Triphosphate; Humans; Inhibitor of Differentiation Protein 1; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Phenotype; Phosphorylation; Plant Extracts; Scutellaria; Shelterin Complex; Telomere-Binding Proteins

Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark

Topics: Algorithms; Animals; Biosensing Techniques; Cell Adhesion; Computer Simulation; Fluorescence Resonance Energy Transfer; Guanosine Triphosphate; Humans; Imaging, Three-Dimensional; Intestines; Intravital Microscopy; Mice; Microscopy, Fluorescence; Models, Biological; Motion; Neoplasm Metastasis; Neuropeptides; Pancreatic Neoplasms; rac1 GTP-Binding Protein; Skin; Software; src-Family Kinases

GTP-binding proteins play important roles in many essential biological processes, including cell signaling, trafficking, and protein synthesis. To assess quantitatively these proteins at the whole proteome level, we developed a high-throughput targeted proteomic method based on the use of isotope-coded GTP probes and multiple-reaction monitoring (MRM) analysis. Targeted proteins were labeled with desthiobiotin-GTP probes, digested with trypsin, and the ensuing desthiobiotin-conjugated peptides were enriched with streptavidin beads for LC-MS/MS analysis. We also established a Skyline MRM library based on shotgun proteomic data acquired for 12 different human cell lines. The library contained 605 tryptic peptides derived from 217 GTP-binding proteins, representing approximately 60% of the annotated human GTP-binding proteome. By using this library, in conjunction with isotope-coded GTP probes and scheduled LC-MRM analysis, we investigated the differential expression of GTP-binding proteins in a pair of primary/metastatic colon cancer cell lines (SW480 and SW620). We were able to quantify 97 GTP-binding proteins, and we further validated the differential expression of several GTP-binding proteins by Western blot analysis. Together, we developed a facile targeted quantitative proteomic method for the high-throughput analysis of GTP-binding proteins and applied the method for probing the altered expression of these proteins involved in colon cancer metastasis.

Topics: Cell Line, Tumor; Colonic Neoplasms; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Isotope Labeling; Neoplasm Metastasis; Proteomics

Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF). Accordingly, downregulation of MITF induces invasion in melanoma cells; however, little is known about the underlying mechanisms. Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C. Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion. We further demonstrate that the gene for guanosine monophosphate reductase (GMPR) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells. Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity and lung colonization. Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAF

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Disease Progression; Ectopic Gene Expression; Extracellular Matrix; Female; Gene Expression Regulation, Neoplastic; GMP Reductase; Guanosine Triphosphate; Heterografts; Humans; Intracellular Space; Melanocytes; Melanoma; Melanoma, Experimental; Mice; Microphthalmia-Associated Transcription Factor; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; rho GTP-Binding Proteins

Cdc42 (cell division control protein 42) and Rac1 (Ras-related C3 botulinum toxin substrate 1) are attractive therapeutic targets in ovarian cancer based on established importance in tumor cell migration, adhesion, and invasion. Despite a predicted benefit, targeting GTPases has not yet been translated to clinical practice. We previously established that Cdc42 and constitutively active Rac1b are overexpressed in primary ovarian tumor tissues. Through high-throughput screening and computational shape homology approaches, we identified R-ketorolac as a Cdc42 and Rac1 inhibitor, distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. In the present study, we establish R-ketorolac as an allosteric inhibitor of Cdc42 and Rac1. Cell-based assays validate R-ketorolac activity against Cdc42 and Rac1. Studies on immortalized human ovarian adenocarcinoma cells (SKOV3ip) and primary patient-derived ovarian cancer cells show that R-ketorolac is a robust inhibitor of growth factor or serum-dependent Cdc42 and Rac1 activation with a potency and cellular efficacy similar to small-molecule inhibitors of Cdc42 (CID2950007/ML141) and Rac1 (NSC23766). Furthermore, GTPase inhibition by R-ketorolac reduces downstream p21-activated kinases (PAK1/PAK2) effector activation by >80%. Multiple assays of cell behavior using SKOV3ip and primary patient-derived ovarian cancer cells show that R-ketorolac significantly inhibits cell adhesion, migration, and invasion. In summary, we provide evidence for R-ketorolac as a direct inhibitor of Cdc42 and Rac1 that is capable of modulating downstream GTPase-dependent, physiologic responses, which are critical to tumor metastasis. Our findings demonstrate the selective inhibition of Cdc42 and Rac1 GTPases by an FDA-approved drug, racemic ketorolac, that can be used in humans.

Topics: Allosteric Regulation; Aminoquinolines; Antineoplastic Agents; Carcinoma, Ovarian Epithelial; cdc42 GTP-Binding Protein; Cell Adhesion; Cell Line, Tumor; Cell Movement; Dose-Response Relationship, Drug; Female; Guanosine Triphosphate; Humans; Ketorolac; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Protein Binding; Pseudopodia; Pyrimidines; rac1 GTP-Binding Protein; Signal Transduction

In the present study we determined the relative contribution of two processes to breast cancer progression: (1) Intrinsic events, such as activation of the Ras pathway and down-regulation of p53; (2) The inflammatory cytokines TNFα and IL-1β, shown in our published studies to be highly expressed in tumors of >80% of breast cancer patients with recurrent disease.. Using MCF-7 human breast tumor cells originally expressing WT-Ras and WT-p53, we determined the impact of the above-mentioned elements and cooperativity between them on the expression of CXCL8 (ELISA, qRT-PCR), a member of a "cancer-related chemokine cluster" that we have previously identified. Then, we determined the mechanisms involved (Ras-binding-domain assays, Western blot, luciferase), and tested the impact of Ras + TNFα on angiogenicity (chorioallantoic membrane assays) and on tumor growth at the mammary fat pad of mice and on metastasis, in vivo.. Using RasG12V that recapitulates multiple stimulations induced by receptor tyrosine kinases, we found that RasG12V alone induced CXCL8 expression at the mRNA and protein levels, whereas down-regulation of p53 did not. TNFα and IL-1β potently induced CXCL8 expression and synergized with RasG12V, together leading to amplified CXCL8 expression. Testing the impact of WT-Ras, which is the common form in breast cancer patients, we found that WT-Ras was not active in promoting CXCL8; however, TNFα has induced the activation of WT-Ras: joining these two elements has led to cooperative induction of CXCL8 expression, via the activation of MEK, NF-κB and AP-1. Importantly, TNFα has led to increased expression of WT-Ras in an active GTP-bound form, with properties similar to those of RasG12V. Jointly, TNFα + Ras activities have given rise to increased angiogenesis and to elevated tumor cell dissemination to lymph nodes.. TNFα cooperates with Ras in promoting the metastatic phenotype of MCF-7 breast tumor cells, and turns WT-Ras into a tumor-supporting entity. Thus, in breast cancer patients the cytokine may rescue the pro-cancerous potential of WT-Ras, and together these two elements may lead to a more aggressive disease. These findings have clinical relevance, suggesting that we need to consider new therapeutic regimens that inhibit Ras and TNFα, in breast cancer patients.

Topics: Animals; Cell Line, Tumor; Chick Embryo; Female; Gene Expression Regulation, Neoplastic; Guanosine Triphosphate; Humans; Interleukin-1beta; Interleukin-8; MAP Kinase Signaling System; MCF-7 Cells; Mice; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; NF-kappa B; Protein Binding; Protein Interaction Domains and Motifs; Proto-Oncogene Proteins p21(ras); Signal Transduction; Transcription Factor AP-1; Transcription, Genetic; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

NRAS mutation at codons 12, 13, or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an NrasQ61R knock-in allele to similarly designed KrasG12D and NrasG12D alleles. With concomitant p16INK4a inactivation, KrasG12D or NrasQ61R expression efficiently promoted melanoma in vivo, whereas NrasG12D did not. In addition, NrasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of NrasQ61R and NrasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, NrasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D. This work identifies a faithful model of human NRAS-mutant melanoma, and suggests that the increased melanomagenecity of NrasQ61R over NrasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways.. This work explains the curious predominance in human melanoma of mutations of codon 61 of NRAS over other oncogenic NRAS mutations. Using conditional "knock-in" mouse models, we show that physiologic expression of NRASQ61R, but not NRASG12D, drives melanoma formation.

Topics: Alleles; AMP-Activated Protein Kinase Kinases; Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Codon; Extracellular Signal-Regulated MAP Kinases; Gene Deletion; Gene Order; Genes, ras; Genetic Loci; Genotype; Guanosine Triphosphate; Humans; Melanoma; Mice; Mitogen-Activated Protein Kinases; Mutation; Neoplasm Metastasis; Oncogene Proteins, Fusion; Phosphatidylinositol 3-Kinases; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins B-raf; Tumor Burden

Protein kinase C (PKC) ε, a key signaling transducer implicated in mitogenesis, survival, and cancer progression, is overexpressed in human primary non-small cell lung cancer (NSCLC). The role of PKCε in lung cancer metastasis has not yet been established.. Here we show that RNAi-mediated knockdown of PKCε in H358, H1299, H322, and A549 NSCLC impairs activation of the small GTPase Rac1 in response to phorbol 12-myristate 13-acetate (PMA), serum, or epidermal growth factor (EGF). PKCε depletion markedly impaired the ability of NSCLC cells to form membrane ruffles and migrate. Similar results were observed by pharmacological inhibition of PKCε with εV1-2, a specific PKCε inhibitor. PKCε was also required for invasiveness of NSCLC cells and modulated the secretion of extracellular matrix proteases and protease inhibitors. Finally, we found that PKCε-depleted NSCLC cells fail to disseminate to lungs in a mouse model of metastasis.. Our results implicate PKCε as a key mediator of Rac signaling and motility of lung cancer cells, highlighting its potential as a therapeutic target.

Topics: Animals; Carcinoma; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Disease Progression; Enzyme Activation; Extracellular Matrix; Gene Expression Regulation, Neoplastic; Guanosine Triphosphate; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Metastasis; Protein Kinase C-epsilon; rac GTP-Binding Proteins; RNA Interference; Signal Transduction

The cancer stem cell (CSC) theory predicts that a small fraction of cancer cells possess unique self-renewal activity and mediate tumor initiation and propagation. However, the molecular mechanisms involved in CSC regulation remains unclear, impinging on effective targeting of CSCs in cancer therapy. Here we have investigated the hypothesis that Rac1, a Rho GTPase implicated in cancer cell proliferation and invasion, is critical for tumor initiation and metastasis of human non-small cell lung adenocarcinoma (NSCLA). Rac1 knockdown by shRNA suppressed the tumorigenic activities of human NSCLA cell lines and primary patient NSCLA specimens, including effects on invasion, proliferation, anchorage-independent growth, sphere formation and lung colonization. Isolated side population (SP) cells representing putative CSCs from human NSCLA cells contained elevated levels of Rac1-GTP, enhanced in vitro migration, invasion, increased in vivo tumor initiating and lung colonizing activities in xenografted mice. However, CSC activity was also detected within the non-SP population, suggesting the importance of therapeutic targeting of all cells within a tumor. Further, pharmacological or shRNA targeting of Rac1 inhibited the tumorigenic activities of both SP and non-SP NSCLA cells. These studies indicate that Rac1 represents a useful target in NSCLA, and its blockade may have therapeutic value in suppressing CSC proliferation and metastasis.

Topics: Adenocarcinoma; Animals; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Knockdown Techniques; Guanosine Triphosphate; Humans; Lung; Lung Neoplasms; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplastic Stem Cells; rac1 GTP-Binding Protein; RNA, Small Interfering; Side-Population Cells

CD97, an adhesion-linked G-protein-coupled receptor (GPCR), is induced in multiple epithelial cancer lineages. We address here the signaling properties and the functional significance of CD97 expression in prostate cancer. Our findings show that CD97 signals through Gα12/13 to increase RHO-GTP levels. CD97 functioned to mediate invasion in prostate cancer cells, at least in part, by associating with lysophosphatidic acid receptor 1 (LPAR1), leading to enhanced LPA-dependent RHO and extracellular signal-regulated kinase activation. Consistent with its role in invasion, depletion of CD97 in PC3 cells resulted in decreased bone metastasis without affecting subcutaneous tumor growth. Furthermore, CD97 heterodimerized and functionally synergized with LPAR1, a GPCR implicated in cancer progression. We also found that CD97 and LPAR expression were significantly correlated in clinical prostate cancer specimens. Taken together, these findings support the investigation of CD97 as a potential therapeutic cancer target.

Topics: Antigens, CD; Cell Line, Tumor; Disease Progression; Extracellular Signal-Regulated MAP Kinases; GTP-Binding Protein alpha Subunits, G12-G13; Guanosine Triphosphate; Humans; Lysophospholipids; Male; Neoplasm Invasiveness; Neoplasm Metastasis; Prostatic Neoplasms; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; rhoA GTP-Binding Protein; Signal Transduction

In addition to their role in nucleotide homeostasis, members of the Nucleoside Diphosphate Kinase (NDPK) family have been implicated in tumor metastasis, cell migration and vesicle trafficking. Although its role in most cases depends on nucleotide catalysis, a precise understanding of how the catalytic activity of NDPK supports its function in diverse processes is lacking. Here we report that wild type, but not catalytically inactive (H118C) NDPKB, undergoes dynamic self-assembly into ordered 20-25 nm diameter filaments in vitro. Self-assembly is nucleoside triphosphate dependent, GTP being most effective at promoting polymer formation. In addition, polymerization appears to depend on formation of the phosphoryl-Histidine intermediate of the enzyme, suggesting a previously unappreciated conformational change in NDPK during its catalytic cycle. We hypothesize that the observed nucleotide-dependent self-assembly property of NDPKB may reflect a key feature of NDPK enzymes that enables their function in diverse processes.

Topics: Biological Transport; Cell Movement; Guanosine Triphosphate; Homeostasis; Humans; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; NM23 Nucleoside Diphosphate Kinases; Protein Structure, Quaternary

Autocrine motility factor (AMF), which is identical to phosphohexose isomerase (PHI)/glucose-6-phosphate isomerase (GPI), a ubiquitous enzyme essential for glycolysis, neuroleukin (NLK), a neurotrophic growth factor, and maturation factor (MF) mediating the differentiation of human myeloid cells, enhances the motility and metastatic ability of tumor cells. AMF/PHI activity is elevated in the serum or urine in patients with malignant tumors. Here, we constructed an amf/phi/nlk/mf gene using adenovirus vector and transfected into two tumor cell lines. Overexpression of AMF/PHI/NLK/MF enhanced AMF secretion into the culture media in both tumor cell lines. However, upregulation of motility and metastatic ability was found only in metastatic fibrosarcoma cells expressing an AMF receptor, gp78, and was not found in gp78-undetectable osteosarcoma cells. Thus, not only serum AMF activity but also gp78-expression in tumor cells may be required for metastasis-related motility induction. With the use of microarray analyses, we detected two augmented genes, rho GDP dissociation inhibitor beta and kinesin motor 3A, as well as AMF itself. The RNA message and protein expression of these two molecules was confirmed to be upregulated, suggesting a possible association with AMF-induced signaling for cell motility and metastasis.

Topics: Adenoviridae; Animals; Cell Line, Tumor; Cell Movement; Glucose-6-Phosphate Isomerase; Guanine Nucleotide Dissociation Inhibitors; Guanosine Triphosphate; Kinesins; Mice; Neoplasm Metastasis; Oligonucleotide Array Sequence Analysis; Signal Transduction; Transfection

Phospholipase D (PLD) is a highly regulated enzyme involved in lipid-mediated signal transduction processes affecting vesicular trafficking and cytoskeletal reorganization. It is regulated by protein kinase C, adenosine diphosphate (ADP)-ribosylation factors and Rho family proteins, and both protein kinase C and Rho family proteins have been implicated in the metastatic potential of melanoma. We analysed PLD in four human melanoma cell lines and in primary human melanocytes. Melanoma cell lines showed phosphatidylcholine-hydrolysing, phosphatidylinositol 4,5-bisphosphate-dependent PLD activity, which was activated by phorbol ester and a non-hydrolysable guanosine triphosphate (GTP) analogue in a dose-dependent and synergistic manner, whereas primary melanocytes exhibited only low PLD activity compared with the melanoma cell lines. As determined by reverse transcription polymerase chain reaction, both splicing variants of PLD1, PLD1a and PLD1b, and the isoenzyme PLD2, are expressed in melanoma cells and melanocytes. Western blot analysis showed that PLD1 expression was low in primary melanocytes in contrast to melanoma cells, which is in agreement with our finding of low activity. Interestingly, Rho protein mRNA was elevated in all melanoma cell lines. We conclude that in human melanoma cells, the PLD activity that is stimulated by phorbol ester requires ADP-ribosylation factor, protein kinase C and Rho proteins for full activity, and most probably represents the isoenzyme PLD1.

Topics: Adenosine Diphosphate; ADP-Ribosylation Factors; Alternative Splicing; Blotting, Western; Cell Division; Cell Line, Tumor; Cells, Cultured; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Activation; Gene Expression Regulation, Enzymologic; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Immunoblotting; Melanocytes; Melanoma; Neoplasm Metastasis; Oleic Acid; Phorbol Esters; Phospholipase D; Protein Isoforms; Protein Kinase C; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; RNA; RNA, Messenger

Endotoxin/lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, is a potent inflammatory stimulus. We previously reported that LPS increased the growth of experimental metastases in a murine tumor model. Here, we examined the effect of LPS exposure on key determinants of metastasis-angiogenesis, tumor cell invasion, vascular permeability, nitric oxide synthase (NOS) and matrix metalloproteinase 2 (MMP2) expression. BALB/c mice bearing 4T1 lung metastases were given an intraperitoneal (i.p.) injection of 10 microg LPS or saline. LPS exposure resulted in increased lung weight and incidence of pleural lesions. LPS increased angiogenesis both in vivo and in vitro. Vascular permeability in lung tissue was increased 18 hr after LPS injection. LPS increased inducible nitric oxide synthase (iNOS) and MMP2 expression in lung tumor nodules. 4T1 cells transfected with green fluorescent protein (4T1-GFP) were injected via lateral tail vein. LPS exposure resulted in increased numbers of 4T1-GFP cells in mouse lung tissue compared to saline controls, an effect blocked by the competitive NOS inhibitor, N(G) methyl-L-arginine (NMA). LPS-induced growth and metastasis of 4T1 experimental lung metastases is associated with increased angiogenesis, vascular permeability and tumor cell invasion/migration with iNOS expression implicated in LPS-induced metastasis.

Topics: Animals; Cell Division; Cell Movement; Disease Models, Animal; Endothelial Growth Factors; Female; Guanosine Triphosphate; Lipopolysaccharides; Lung Neoplasms; Lymphokines; Mammary Neoplasms, Experimental; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasm Metastasis; Neovascularization, Pathologic; Nitric Oxide Synthase; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Processes like cell proliferation, differentiation, and tumor metastasis require a flexible adaptation of cell shape and cell plasticity. A regulator of cell structure and shape is the centrosome and its associated microtubules. Recently, oncogenes like p53, pRB, and the tumor suppressor BRCA1 have been characterized as members of the centrosome. In this communication, we identified rat Nm23-R1/NDPKbeta, a homologue of the human tumor metastasis suppressor Nm23-H1 and a regulator of cell proliferation and differentiation, as a component of the centrosomal complex. We used confocal laser scanning microscopy on different cell types and biochemical analysis of purified centrosomes to demonstrate that Nm23-R1 is located in the centrosome of dividing and nondividing cells. We also showed that the centrosomal enzyme is catalytically active and able to transfer the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. In addition, Nm23-R1 coimmunoprecipitated with gamma-tubulin, a core centrosomal protein essential for microtubule nucleation. In addition, human Nm23-R1/-H1 was also shown to be present in the centrosome of different human and rat cell types, demonstrating that the presence of Nm23-H1 homologues in the latter organelle is a general event.

Topics: Adenosine Triphosphate; Adrenergic beta-Agonists; Animals; Cell Differentiation; Cell Division; Cells, Cultured; Centrosome; Glioma; Guanosine Triphosphate; Immunohistochemistry; Isoenzymes; Microtubules; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Precipitin Tests; Rats; Recombinant Proteins; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tubulin

Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP.Rad was also reconverted to GTP.Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation.

Topics: Animals; Diabetes Mellitus; DNA; Enzyme Activation; Genes, Tumor Suppressor; Glucose; GTP Phosphohydrolases; Guanosine Triphosphate; Humans; Immediate-Early Proteins; Models, Biological; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Protein Binding; ras Proteins; Rats; Rats, Sprague-Dawley; Transcription Factors

We have introduced point mutations in v-rasH to study their effects on biochemical and biological properties of the ras-encoded protein p21. Several of these mutant proteins do not bind GTP and thus lack GTPase activity, while others were shown to have their GTP binding reduced. We have introduced these ras mutants into NIH 3T3 fibroblastoid cells to study major parameters of clinical importance which are associated with neoplastic transformation, particularly MHC expression in cells, metastasis and tumorigenesis in both nude mice and immune competent mice. Our data show that certain mutations in v-ras differentially affect the expression of the transformed phenotype. Mutant ras molecules deficient in GTP binding fail to generate rapidly progressing tumors in immune competent mice, and not all morphologically transformed cells were capable of experimental metastasis. Cells transformed by certain v-ras mutants form tumors in immunocompetent mice and show reduced expression of MHC class-I antigens. Other cells are morphologically transformed and tumorigenic in athymic nude mice, but fail to form tumors in normal mice and show levels of MHC class-I antigen expression similar to non-transformed 3T3 cells. The inverse relationship between MHC class-I-antigen expression and the degree of transformation in fibroblastoid cells suggests that the ras gene product could be involved in regulating MHC expression.

Topics: Animals; Blotting, Southern; Cell Line; Cell Transformation, Neoplastic; Clone Cells; DNA, Neoplasm; Fibroblasts; Gene Expression Regulation, Neoplastic; Genes, MHC Class I; Genes, ras; GTP Phosphohydrolases; Guanosine Triphosphate; Major Histocompatibility Complex; Mice; Mice, Inbred BALB C; Mice, Nude; Mutagenesis; Neoplasm Metastasis; Neoplasms, Experimental; Proto-Oncogene Proteins p21(ras); Transfection